Secretion of insulin from beta-cells of pancreatic islets is biphasic and pulsatile. Loss of first phase secretion and steady-state plasma insulin oscillations are an early development in Type 2 diabetes mellitus. Despite extensive study of the signaling cascades underlying glucose-stimulated insulin secretion, it remains unclear how oscillations in insulin secretion arise. Our overall hypothesis is that slow oscillations in islet metabolism and insulin secretion reflect slow oscillations in glycolyis. Based on this hypothesis, a computational model has been developed in which slow glycolytic oscillations mediated by phosphofructokinase-1 (PFK1) interact with fast oscillations arising from membrane electrical activity and Ca2+ (the 'Dual Oscillator Model', or DOM). Although the DOM can uniquely account for the diversity of oscillatory patterns observed in islets in vitro and in vivo, direct evidence for beta-cell glycolytic oscillations is lacking, leaving the open question of whether metabolic oscillations are intrinsic, as the DOM predicts, or driven by Ca2+ oscillations, as proposed in competing models. To test the predictions of the DOM at the level of metabolic activity, we propose to examine whether glucokinase and phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB), which are regulators of PFK1, represent oscillatory parameters that modulate metabolism-secretion coupling in islets. Dynamic FRET imaging using novel metabolic sensors will be combined with patch-clamp electrophysiology, mathematical modeling, and optical measurements of Ca2+ and NAD(P)H, to address the following Specific Aims: 1. Define the spatial and temporal properties of glucokinase activation, including regulation via its binding partner PFKFB, and assess their respective contributions to the modulation of metabolic oscillations. 2. Modify the Dual Oscillator Model to incorporate changes in glycolytic flux and islet oscillatory behavior induced by glucokinase/PFKFB interaction and fructose-2,6-bisphosphate production by PFKFB. 3. Determine whether Ca2+ drives changes in metabolism, metabolism drives Ca2+, or both occur. These studies are designed to provide novel insights into the origin of oscillations in insulin secretion. Insulin oscillations are suppressed in patients with Type 2 diabetes mellitus and their near relatives. Understanding these defects may lead to new approaches for the diagnosis and treatment of Type 2 diabetes mellitus and related metabolic diseases. PUBLIC HEALTH RELEVANCE: These studies are designed to provide novel insights into the origin of oscillations in insulin secretion. Insulin oscillations are suppressed in patients with Type 2 diabetes mellitus and their near relatives. Understanding these defects may lead to new approaches for the diagnosis and treatment of Type 2 diabetes mellitus and related metabolic diseases.